Field of the Invention
Embodiments of the invention are directed to the field of biology. More particularly, embodiments of the invention are directed to a system for use in processing formalin-fixed paraffin-embedded (FFPE) tissue samples for molecular biology studies, and applications thereof.
Description of Related Art
An aspect of histology relies on preserving tissue from decay for later examination or research. Such tissues are often preserved by subjecting them to a chemical (often a formalin solution) that prevents decay both from autolysis and/or putrefaction of the tissue. Chemical fixatives may also preserve the tissue's structure by introducing chemical cross-linking among the amine groups of the tissue's proteins. The same tissues are then often embedded in a hydrocarbon matrix (often paraffin wax) to more conveniently store them in a solid phase and to more reliably remove thin slices of the fixed tissue for microscopic investigation. The tissues thus preserved are commonly termed formalin fixed paraffin embedded (FFPE). With the introduction of molecular biology, the FFPE tissues are not only destined for microscopic analysis but they may often be of interest for investigation of the genetic material also preserved in the tissue. In such cases the paraffin wax needs to be removed from the tissue and the cross-linking resulting from the fixative needs to be reversed in order to separate the genetic material from the FFPE tissue and render it compatible with the chemistry required for molecular biology investigation of the tissue. FFPE tissue samples are traditionally dewaxed from the paraffin wax by placing the embedded tissue sample in a xylene solution. The paraffin wax then dissolves into the xylene, whereafter the tissue sample is removed from the xylene and rehydrated with an ethanol/xylene mixture in series dilution processes. Alternatively, xylene may be sprayed onto a tissue sample and the dissolved paraffin “washes away” as new xylene is applied to the tissue sample. While xylene is an efficient solvent for sample de-waxing, its introduction into tissue processing steps makes the chemistry used in molecular biology difficult; it is also a hazardous chemical for lab technicians to handle (generally requiring a ventilated hood and special waste disposal means), and its organic nature makes many aqueous buffer steps automation a challenge. A system that can accomplish the removal of the paraffin wax without the use of xylene is advantageous. Furthermore, if the system were an aqueous system compatible with subsequent molecular biology processes for nucleic acid assays, the technique would be a significant improvement over the use of xylene.
Tissue samples embedded in paraffin wax are often “fixed” chemically prior to embedding. The fixing is accomplished by formation of cross-linking methylene bridges between amino groups. This polymeric network structure results in low permeability of macromolecules in a protein's backbone but the structural features of the protein molecules are well preserved. The chemical cross-linking renders the tissue immune from decay. Therefore it can then be stored for long periods in ambient conditions. In order for the fixed tissue to yield nucleic acids suitable for molecular biological investigation the chemically induced cross-linking needs to be reversed so that the nucleic acids can be purified out of the tissue sample. The de-cross-linking can be achieved through subjecting the tissue sample (once the paraffin has been removed) to a suitable buffer which may also contain a detergent or a surfactant and suitably elevated temperatures to reverse the cross-linking. Therefore a system and method that can easily remove the paraffin wax from the tissue and then proceed to de-crosslink the macromolecules thus rendering the tissue sample in a condition compatible with classical aqueous based sample preparation, purification and nucleic acid amplification would be very useful and, even more advantageous if the system can be performed automatically.